Abstract:

The present invention relates to methods of inducing protein folding using
light illumination. More specifically the invention relates to
shape-reconstruction analysis applied to small angle neutron scattering
(SANS) data that is used to determine the structure of partially-folded
proteins in non-native conformations and supramolecular complexes
undergoing self- or hetero-association in solution as a result of partial
unfolding with a photoresponsive surfactant.

Claims:

1. A method for determining the structure of partially-folded proteins in
non-native conformations and supramolecular complexes undergoing self- or
hetero-association in solution comprising:a) allowing proteins to
interact with photosensitive surfactants containing an azobenzene
group;b) exposing said proteins and surfactants to light illumination;c)
determining the small-angle neutron scattering (SANS) of said proteins;
andd) applying SANS data to shape-reconstruction analysis,wherein said
surfactant undergoes photoisomerization upon exposure to light.

2. The method according to claim 1, wherein said protein is an
amyloid-forming protein.

3. The method according to claim 1, wherein said protein is
6-chymotrypsin.

4. The method according to claim 1, wherein said surfactant is
azobenzene-trimethylammonium bromide.

5. The method according to claim 1, wherein said non-native conformations
are prefibrillar intermediates.

6. The method according to claim 5, wherein said prefibrillar intermediate
is a protofibril, a protofilament, or a fibril intermediate.

7. The method according to claim 1, wherein said light is visible or UV
light.

8. A method of using light illumination to induce photoreversible changes
in both the secondary and tertiary structure of proteins comprising:a)
allowing said proteins to interact with photosensitive surfactants
containing an azobenzene group; andb) exposing said proteins and said
surfactants to light illumination, wherein said surfactant undergoes
photoisomerization upon exposure to light and wherein the isomerization
reverses when the light exposure is removed.

9. The method according to claim 8, wherein said protein is an
amyloid-forming protein.

10. The method according to claim 8, wherein said protein is {acute over
(α)}-chymotrypsin.

11. The method according to claim 8, wherein said surfactant is
azobenzene-trimethylammonium bromide.

12. The method according to claim 8, wherein said non-native conformations
are prefibrillar intermediates.

13. The method according to claim 12, wherein said prefibrillar
intermediate is a protofibril, a protofilament, or a fibril intermediate.

14. The method according to claim 8, wherein said light is visible or UV
light.

15. A method of generating conformations of partially-folded proteins in
non-native conformations in solution comprising:a) determining
small-angle neutron scattering (SANS) of said proteins intermediates;
andb) applying SANS data to the shape-reconstruction algorithm
GA_STUCT,wherein the weight-average molecular weight (Mw) is
calculated from the equation M W = 1000 I ( 0 ) N A c
υ _ 2 ( ρ P - ρ S ) 2 ,
##EQU00005## where ρS and ρP are the scattering length densities
of the solvent (6.36.times.10.sup.10 cm-2) and protein
(3.23.times.10.sup.10 cm-2), respectively, c is the protein
concentration (11.6 mg/mL at pH 3 and 11.4 mg/mL at pH 7), and υ
is the protein specific volume (0.734 cm3/g,I(0)-values were
determined from Guinier plots usingI(Q)=I(0)exp(-Q2Rg2/3),
where Rg is the radius of gyration,pair distance distribution
functions were calculated from the SANS data according to the equation
I ( Q ) = 4 π ∫ 0 D max P ( r ) sin
( Q r ) Q r r , ##EQU00006## where
P(r) is related to the probability of two scattering centers (nuclei for
SANS) being a distance r+dr apart, and Dmax is the maximum distance
between scattering centers within the protein or protein oligomer, and
I(0)-values were then obtained from the PDDFs through
I(0)=4.pi.∫.sub.0.sup.D.sup.maxP(r)dr.

Description:

FIELD OF THE INVENTION

[0002]The present invention relates to the use of photo-responsive
surfactants to reversibly control protein aggregation with light
illumination.

BACKGROUND OF THE INVENTION

[0003]Proteins interact with a variety of molecules during the course of
activity, ranging from small ions and ligands to other proteins through
either heterogeneous or homogenous association. Indeed, the dynamic and
multifarious response of proteins to these interactions is utilized to
stimulate or regulate virtually every biological process. In some cases,
however, protein interactions can result in unwanted or deleterious
effects, such as protein-protein associations leading to amyloid fibril
formation. The most well-known example of this process involves the
aggregation of the amyloid-beta (Aβ) peptide fragments Aβ40 and
Aβ42 implicated in Alzheimer's disease, although amyloid fibrils
have been observed in an array of proteins largely independent of the
native secondary structure, (1) including ribonuclease A, (2) an SH3
domain, lysozyme, (3) insulin, (4) and α-chymotrypsin. (5) This
process is generally believed to result from the formation of unstable
slightly-unfolded conformations, leading to a cascading aggregation
process from monomers to oligomers (unstructured aggregates of typically
multiples of six molecules in the case of Aβ42(6)) to protofibrils
(structured aggregates exhibiting β-sheet structure) to
protofilaments (elongated aggregates ˜2-5 nm in diameter) to
fibrils (2-6 entwined protofilaments). (1) Perhaps most importantly, the
prefibrillar intermediates (i.e., oligomers and protofibrils), which can
induce cognitive impairment, have become increasingly viewed as the
primary pathogenic species. (1, 7)

[0004]To date, however, the solution structure of these important
intermediate species remains unknown as the two preferred methods to
determine protein structure, namely X-ray crystallography and solution
NMR, are generally limited to the study of native proteins in the solid
state or relatively small protein assemblies, respectively, since protein
crystallization is often supplanted by unwanted aggregation and
crystal-packing constraints largely dominate protein orientations in
multi-molecular complexes. Thus, the development of novel structural
characterization methods capable of examining partially-folded proteins
in non-native conformations and supramolecular complexes undergoing self-
or hetero-association is highly desired. For example, through the use of
small-angle neutron scattering (SANS), the low-resolution in vitro
structures of Aβ40 protofibrils were found to by cylindrical with
cross-sectional radii of 24 Å and 110 Å long, (8) while through
AFM and TEM measurements a variety of protofibril arrangements have been
observed, including twisted chains 2-5 nm in diameter. However, without
higher-resolution conformations, and due to potential influences of
surface interactions, the precise nature of protofibrils conformation in
solution remains unknown. As a result, to properly investigate
intermediate conformations in an amyloid protein necessitates two
complementary approaches: (1) a means to induce changes in protein
folding and, hence, association in a controlled and preferably reversible
manner, and (2) a method to determine the conformation of non-native and
associated proteins at relatively high resolution.

[0005]Recently, the inventors have shown that light illumination can be
used to induce photoreversible changes in both the secondary (9) and
tertiary (10, 11) structure of proteins. This method utilizes the
interaction of proteins with photosensitive "azoTAB" surfactants
containing an azobenzene group that undergoes a trans (relatively
hydrophobic) to cis (relatively hydrophilic) photoisomerization upon
exposure to visible (434 nm) or LV (350 nm) light illumination,
respectively. Hence, light can be used to reversibly bind the surfactant
to the hydrophobic domains of proteins, leading to photocontrol of
protein folding. Furthermore, the inventors have applied small-angle
neutron scattering (SANS) to study the in vitro structure of the
non-native protein conformations that form in response to photosurfactant
and light. Small-angle neutron and X-ray scattering have been used for
several decades to investigate the structure of soluble proteins in
solution (12-15) and membrane proteins in surfactant assemblies (16, 17).
The obtained structures have typically been low-resolution, however, a
consequence of modeling proteins with a single dimension (radius of
gyration) or as ellipsoids (axial radii). These procedures, although
convenient, belie the wealth of structural information contained within
the measured scattering intensity. From the range of momentum vectors
Q=4πλ-1 sin(θ/2), where λ is the neutron
wavelength (6 Å) and θ is the scattering angle, in a typical
SANS experiment (Q=0.005-0.5 Å-1), it can be seen that the data
span length scales (L=2π/Q) ranging from 12.5-1250 Å, ideal for
protein conformational studies. Indeed, application of
shape-reconstruction techniques such as the ab initio methods of GASBOR
(14) and GA_STRUCT (15) reveals a high degree of similarity between the
native structure in solution (SANS) and in the solid state (X-ray
crystallography), a seemingly general property of soluble proteins. (12)

SUMMARY OF THE INVENTION

[0006]In the present invention, the ability to photo-initiate changes in
protein quaternary structure through photoreversible control of
α-chymotrypsin self-association is demonstrated. Native
α-chymotrypsin is well-known to self-associate through either a
monomer-dimer (pH 3) or monomer-hexamer (pH 7) equilibrium, while the
addition of trifluoroethanol, a solvent known to induce partially-folded
structures (18, 19) has been reported to result in α-chymotrypsin
amyloid-fibril formation. (5) Mixing α-chymotrypsin with the
photoresponsive azoTAB surfactant is found to result in partial unfolding
of the protein, giving rise to changes in both the degree and type of
self-association. Shape-reconstruction analysis applied to SANS data
allows determination of the in vitro conformation of α-chymotrypsin
oligomers. In the presence of azoTAB under visible light, native
oligomers (dimer or compact hexamers) are converted to expanded
corkscrew-like hexamers, while upon UV-light illumination the hexamers
laterally aggregate, wrapping around each other to form dodecamers with
twisted conformations. FT-IR measurements of the protein secondary
structure reveal that dodecamer formation is accompanied by hydrogen-bond
stabilized intermolecular β sheets, commonly observed in amyloid
fibrils. TEM measurements following incubation further confirm to
formation fibrillar structures, while, photo-reversibility of the
hexamer-to-dodecamer association process is studied with small-angle
X-ray scattering (SAXS) measurements. Together, these results provide
what is believed to be the first direct observation of the mechanism of
formation of the key intermediates in an amyloid-forming protein, which
should provide unique insight into the amyloidosis disease pathway.

[0007]The above-mentioned and other features of this invention and the
manner of obtaining and using them will become more apparent, and will be
best understood, by reference to the following description, taken in
conjunction with the accompanying drawings. The drawings depict only
typical embodiments of the invention and do not therefore limit its
scope.

[0013]FIG. 6. Shape-reconstructions of the oligomer-only SANS data at (a)
pH 3 and (c) pH 7 as a function of azoTAB concentration and light
illumination. Inserts show four views of the shape reconstructions
rotated at 90°, along with low-resolution globular models designed
to mimic the twisted conformations detected in the structures.

[0014]FIG. 7: (a) FT-IR absorbance spectra for pure α-Ch (black) and
mixtures of α-Ch with 9.04 mM azoTAB under visible light (red) and
UV light (blue). (b) FT-IR difference spectra (UV--visible) demonstrating
the effect of light illumination. Also shown are Congo red fluorescence
(c) and apple green birefringence (d) obtained under cross polarizers, as
well as TEM images of a fresh solution (e-f) (pH3, [azoTAB]=4.95 mM) and
an original SANS solution (g) (pH3, [azoTAB]=4.23 mM) after an elapsed
time of approximately one year.

[0016]Shape-reconstruction analysis applied to small angle neutron
scattering (SANS) data was used to determine the in vitro conformations
of α-chymotrypsin oligomers that form as a result of partial
unfolding with a photoresponsive surfactant. In the presence of the
photo-active surfactant under visible light, the native oligomers (dimers
or compact hexamers) rearrange into expanded corkscrew-like hexamers.
Converting the surfactant to the photo-passive form with UV-light
illumination causes the hexamers to laterally aggregate and intertwine
into dodecamers with elongated, twisted conformations containing
cross-sectional dimensions similar to amyloid protofilaments.
Secondary-structure measurements with FT-IR indicate that this
photo-induced hexamer-to-dodecamer association occurs through
intermolecular β sheets stabilized with hydrogen bonds, similar to
amyloid formation. SANS is ideally suited to the study of these
associated intermediates, providing direct observation of the mechanism
of oligomeric formation in an amyloid-forming protein. Combined with
photoreversible hexamer-to-dodecamer associations in the presence of the
photoresponsive surfactant, the present invention should provide insight
into the amyloidosis disease pathway, as well as disease treatment
strategies.

[0017]The following examples are intended to illustrate, but not to limit,
the scope of the invention. While such examples are typical of those that
might be used, other procedures known to those skilled in the art may
alternatively be utilized. Indeed, those of ordinary skill in the art can
readily envision and produce further embodiments, based on the teachings
herein, without undue experimentation.

Experimental Methods

[0018]An azobenzene-trimethylammonium bromide surfactant (azoTAB) of the
form

##STR00001##

similar to surfactants used in previous studies, (10, 11, 20, 21) was
synthesized according to published procedures. (22, 23) When illuminated
with 350-nm UV light, the surfactant undergoes a photoisomerization
predominantly to the cis form (90/10 trans/cis), which can be rapidly
reversed upon exposure to visible light (434 nm, 75/25 trans/cis) or in
the dark in about 24 hours (˜100% trans isomer). (20) For the SAXS
and FT-IR measurements, conversion to the UV-light form was achieved with
the 365-nm line from a 200 W mercury arc lamp (Oriel, model 6283),
isolated with the combination of a 320-nm band-pass filter (Oriel, model
59800) and an IR filter (Oriel, model number 59060). A 400-nm long-pass
filter (Oriel, model 59472) was used to convert back to the visible-light
form. In the SANS experiments, the solutions were exposed to an 84 W
long-wave UV lamp (365 nm, Spectroline, model XX-15A) for at least 30 min
prior to sample collection to convert to the UV-light form, and were
continuously exposed to the same UV light throughout the data collection.

[0020]Small-angle neutron scattering experiments were performed on the
30-m NG3 SANS instruments at NIST. (24) A neutron wavelength of λ=6
Å and a detector offset of 25 cm with two sample-detector distances
of 1.33 and 7.0 m were utilized to achieve a Q range of 0.0048-0.46
Å-1. The net intensities were corrected for the background and
empty cell (pure D2O), accounting for the detector efficiency using
the scattering from an isotropic scatterer (Plexiglas), and converted to
an absolute differential cross section per unit sample volume (in units
of cm-1) using an attenuated empty beam. The data were then
corrected for incoherent scattering by subtracting a constant background.
The shape-reconstruction algorithm GA_STUCT (11) was used to generate
solution conformations, similar to previous studies (10, 11). Beginning
with an initial guess of randomly-distributed scattering centers, the
program rearranges the position of the scattering centers to best fit the
experimental scattering data.

[0021]The weight-average molecular weight (Mw) of each sample was
calculated from the equation

M W = 1000 I ( 0 ) N A c υ _ 2 (
ρ P - ρ S ) 2 , ( 1 ) ##EQU00001##

where ρS and ρP are the scattering length densities of the solvent
(6.36×1010 cm-2) and protein (3.23×1010
cm-2), respectively, c is the protein concentration (11.6 mg/mL at
pH 3 and 11.4 mg/mL at pH 7), and υ is the protein specific
volume (0.734 cm3/g). (25) I(0)-values were determined from Guinier
plots (25) using I(Q)=I(0)exp(-Q2Rg2/3), where Rg is
the radius of gyration. Guinier plots, generally valid for
QRg<1.3, can be influenced by solution structuring due to
intermolecular interactions between charged proteins, which becomes
increasingly important as Q decreases below 1.5/R (10) (or <0.05
Å-1 using an α-Ch radius ˜30 Å). Thus, pair
distance distribution functions were calculated from the SANS data using
the program GNOM (26) according to the equation

where P(r) is related to the probability of two scattering centers (nuclei
for SANS) being a distance r+dr apart, and Dmax is the maximum
distance between scattering centers within the protein or protein
oligomer. I(0)-values were then obtained from the PDDFs through
I(0)=4π∫0D.sup.maxP(r)dr, which has the advantage of
utilizing the entire Q-range to determine I(O), as opposed to just the
low-Q values as in Guinier analysis (27).

[0022]The small-angle X-ray scattering data were measured using the X21
beamline at the National Synchrotron Light Source at the Brookhaven
National Laboratory. (28) The X-ray wavelength was set to 1.24 Å with
a pair of Si(111) monochromator crystals. The sample-to-detector distance
was calibrated to be 1.69 m using a silver behenate standard. To avoid
radiation damage, solutions were continuously passed at a flow rate of 60
μL/min through a 1-mm glass capillary housed within an aluminum block
containing Plexiglas observation windows. (28) The net intensities were
corrected for the background and solvent scattering as well as sample
transmission, and were put on an absolute scale by comparison with a
calibration standard (10 mg/mL BSA (10)).

[0023]Infrared spectra were measured with a Genesis II FT-IR spectrometer
(Mattson Instruments). Solutions were loaded in a demountable liquid cell
equipped with a circulating water jacket (T=20° C.) between a pair
of CaF2 windows using a 50 μm Teflon spacer. A liquid light guide
(Oriel, model no. 77557) was used to directly illuminate the sample with
UV or visible light for 2 hours prior to and during data collection, as
previously described (9). The sample chamber was continuously purged with
dry air to eliminate the influence of water vapor. For each spectrum, a
500-scan interferogram was collected with a 2 cm-1 resolution. The
relatively sharp surfactant peaks at ˜1600 cm-1 were removed
by subtracting the spectra measured for a pure surfactant solution under
otherwise identical conditions, resulting in corrected spectra that were
flat in the region between 2000 and 1750 cm-1. Fourier
self-deconvolution (FSD) was applied to spectra to resolve the
overlapping bands in the Amide I region using a band-narrowing factor
k=2.0 and a full width at half height of 12.6 cm-1. Second
derivative spectra were obtained with the Savitsky-Golay function for a
3rd order polynomial, using a 13 data point window. Difference
spectra were obtained by subtracting the spectra collecting under visible
light from the spectra collecting under UV light illumination. Difference
spectra obtained for pure α-chymotrypsin solutions without
surfactant shown no significant absorbance changes (<1% throughout the
amide I region).

[0025]Transmission electron microscopy was performed on a Philips EM420
TEM operating at 80 kV. A drop of protein solution was placed on a
carbon-coated grid for 10 seconds and then blotted with filter paper,
followed by repeating this procedure with a second drop. The grid was
then placed in a freshly made 1 wt % uranyl acetate solution for 30 s.

Results and Discussion

[0026]SANS data for α-Ch/azoTAB mixtures are shown in FIG. 1 as a
function of pH, surfactant concentration, and light illumination. AzoTAB
undergoes a photoisomerization to the relatively-hydrophilic cis form
when illuminated with 350-nm UV light, which can be reversed back to the
relatively-hydrophobic trans form upon exposure to 434-nm visible light.
(20) In inverse space (e.g., with Q in units of Å-1), the
transitions responsible for SANS intensity changes in FIG. 1 can be
difficult to conceptualize, thus, the real space length scale L
(=2π/Q) is plotted on the upper x-axis. The addition of azoTAB causes
an increase in scattering at low-Q (i.e., L>100 Å), suggesting the
surfactant induces monomer→oligomer associations. UV-light
illumination further enhances this effect, with a shift in the scattering
curves to lower Q indicating greater protein aggregation when the
surfactant is converted to the cis form. Thus, the trans isomer appears
capable of replacing protein-protein interactions with protein-surfactant
interactions. Beyond Q>0.2 Å-1 (L<30 Å, or length
scales less than the protein diameter) the SANS data converge, suggesting
that the individual protein subunits remain relatively intact. However,
at high Q the resolution (˜20 Å) of the SANS data is approached
due to weak sample scattering relative to incoherent scattering from the
protein (0.003 cm-1) and solvent (0.0004 cm-1 for 99.9%
D2O).

[0027]For associating systems, SANS has two advantageous properties.
First, SANS is an absolute technique with the weight-average molecular
weight (Me) of the sample given directly by I(0), the scattering at zero
angle (see also Experimental Methods). Thus, the weight fraction of
protein existing as monomer (x1) and n-mer (xn=1-x1) can
be calculated from Mw=x1Mw,1+(1-x1)Mw,n, where
Mw,1 and Mw,n are the monomer and n-mer molecular weights,
respectively. Second, SANS is additive with the scattering for a mixture
of monomer (1-mer) and n-mer species given by the sum of the
contributions from each oligomer o from 1 to n (29)

I ( Q ) = n p ( o = 1 n N o N F o (
Q ) 2 ) S _ ( Q ) , ##EQU00003##

where

n p = 1 V o = 1 n N o = N V ##EQU00004##

is the total number of particles per volume, No/N is the number
fraction of a given type of oligomer, F0(Q) is the form factor for
that oligomer, and S(Q) is the averaged structure factor related to the
partial structure factors Sij(Q). Hence for a non-interacting
mixture of monomer and a single n-mer, (30) the scattering intensity can
be shown to be I=ν1I1+νnIn, where ν1
and νn are the fractions of protein existing as monomer and n-mer
on a volume basis (not to be confused with the volume fraction in
solution, φ=c υ/1000, where c is the protein concentration in
mg/mL units and υ is the protein specific volume), while I1
and In are the scattering from pure monomer and n-mer, respectively.
(27) Since νi˜xi, assuming that υ is constant
independent of oligomeric state, the total scattering intensity is then
also given by the linear combination I=x1I1+xnIn.
(31) Thus, these two properties of SANS can be utilized to assign the
contributions of the overall scattering to the monomer and n-mer,
followed by shape-reconstruction to determine the in vitro structure of
α-Ch oligomers. In the sections that follow this will first be
illustrated for pure α-Ch, then extended to solutions containing
azoTAB to demonstrate photo-reversible α-Ch association.

Pure Protein Solutions

[0028]α-Ch is well-know to self associate through either a
monomer-dimer (pH 3) or monomer-hexamer (pH 7) equilibrium at low ionic
strength, (32-34) with a reduction in the overall positive charge of the
protein (pI=9.1) with increased pH generally allowing for greater
association. The SANS data for pure α-Ch solutions shown in FIG. 1,
measured at conditions where self-association is expected to be prevalent
(˜10 mg/mL protein), are re-plotted in FIG. 2. The raw data are
largely featureless due to the presence of both monomer and oligomer in
solution, complicating quantitative analysis of the self-association
process. To deconvolute the scattering data, the weight-average molecular
weight (Mw) of each sample was calculated from the scattering at
zero angle, reported as the effective oligomer size
(neffMw/M1) in Table 1. I(0) values determined from both
Guinier plots (25) using I(Q)=I(0)exp(-Q2Rg2/3),
technically valid for QRg<1.3 as discussed below, where Rg
is the radius of gyration (FIGS. 2a and 2c, insets), and from the entire
Q-range using pair distance distribution functions (PDDFs) in FIGS. 2b
and 2d, are generally consistent. Note that the steep upturn in the
Guinier plots at Q<0.01 Å-1 (L>600 Å) could be due to
the presence of a small amount of higher-order aggregates, which due to
the characteristic Q-4 decay would not be expected to influence the
data analyses employed below.

[0029]From the Mw-value determined at each pH, the weight fraction of
monomer and n-mer were calculated. The portion of the scattering
resulting from free monomers, estimated from the monomer PDB file 2CHA
using the program CRYSON (35) and scaled with a monomer concentration of
x1c as shown in FIGS. 2a and 2c, were then subtracted from the
overall scattering to give the oligomer-only SANS data. This procedure
presumes that the structure of the monomer in vitro is well represented
by the native X-ray crystallographic structure, shown to be true for a
range of soluble proteins (12, 36, 37). Compared to the featureless raw
data, the oligomer scattering curve at pH 7 displays a prominent peak at
Q=0.14 Å-1, translating in real space to L ˜45 Å, also
detected as a peak in the corresponding PDDF curve. This dimension
corresponds to a highly probable distance within the protein oligomer,
namely the separation distance between monomers. Peaks in this Q-range
signify well-ordered oligomer conformations and are often used as
qualitative tests for oligomer formation, (38) lending confidence in the
deconvolution procedure. Oligomer peaks become more pronounced with
increasing n-mer size, thus, it is not surprising that the dimer data at
pH 3 do not display this peak.

[0030]Following deconvolution, shape-reconstructions of the oligomer-only
data were performed, conceptually similar to previous studies used to
determine the in vitro structures of partially-folded BSA (10, 21) and
lysozyme (11). The GA_STRUCT program begins with chains of randomly
oriented "scattering centers" (i.e., atomic nuclei for SANS), with a
genetic algorithm consisting of matings, mutations, and extinctions used
to update the shape. (15) Despite this general procedure, the dimer (pH
3) and hexamer (pH 7) structures indeed contain n subunits for each
n-mer, as shown in FIG. 3. Interestingly, the SANS-based in vitro dimer
is not consistent with the "face-to-face" (active site-to-active site)
crystal packing of α-chymotrypsin, and is instead better
represented by the "back-to-side" packing of chymotrypsinogen (2CGA, note
that α-Ch results from the removal of two dipeptides at positions
14-15 and 147-148 in chymotrypsinogen). For example, the maximum
dimension of 6CHA ˜70 Å, while the PDDF in FIG. 2b gives a
Dmax of 90 Å compared to ˜85 Å for 2CGA. This serves
to highlight the influence that crystal-packing constraints can have on
molecular orientations, a significant advantage of SANS in the study of
protein aggregates, and could explain why the role of the active site in
α-Ch association remains unsolved in the literature with different
techniques yielding conflicting results. (32)

[0031]Shape-reconstruction of the pH 7 data reveals the compact,
"W-shaped" hexamer shown in FIG. 3b. The average distance between
nearest-neighbor subunits is 43±5 Å, in agreement with the 0.14
Å-1 peak in FIG. 2c, while the orientation angle between three
successive subunits is 70±10°. The consistency of these values
suggests that specific intermolecular interactions are responsible for
hexamer formation in solution, resulting in the twisted arrangement of
the subunits. Interestingly, the ribbon diagram of a hypothetical hexamer
constructed by continuing the relative orientation of the two
macromolecules in 2CGA (with alternate proteins color-coded blue and
green) exhibits a similar twisted orientation, with all but the final
protein in nearly identical locations. In contrast, the face-to-face
arrangement of 6CHA would not support higher-order association, as
opposed to the "heterologous association" apparently observed in FIG. 3a.
(31) Also shown in the inset of FIG. 3 is the consensus envelope obtained
by docking and averaging ten independent fits of the GA_STRUCT program,
along with the run that statistically produced the worst fit to the data.
Both of these structures agree with the W-shaped hexamer conformation,
demonstrating that the coupled deconvolution/shape-reconstruction
technique can be applied to protein oligomers in solution. Non-native
protein conformations such as partially-folded or associated states
challenge existing crystallographic and NMR methods. However, as
demonstrated in FIG. 3 and in recent studies of photo-controlled protein
folding (10, 11), SANS can provide valuable information on these
important yet understudied class of structures.

α-chymotrypsin/azoTAB solutions

[0032]As discussed above, qualitative assessment of the SANS data in FIG.
1 indicate enhanced α-Ch association with either increased
surfactant concentration or upon converting azoTAB from the trans to the
cis form with UV-light illumination. To quantitatively investigate this
phenomena, Guinier plots of the SANS data for α-Ch/azoTAB solutions
were generated, as shown in FIGS. 4a and 4b. Two unique slopes can be
detected at each condition, the first in the region of Q2<0.002
Å-2 giving the z-average radius of gyration of the mixture, and
the second at Q2˜0.01-0.03 Å-2 with Rg-values
ranging from 17.0-18.2 Å, as shown in Table 1. These latter values
are consistent with the Rg of monomeric α-Ch in the literature
of 16.9 Å (39), thus, indicating a monomer/n-mer equilibrium, (40,
41) similar to the monomer-oligomer equilibrium observed during the early
stages of fibril formation of Aβ proteins. (1, 8) The 7% increase in
Rg with the addition of azoTAB in Table 1 indicates that a slight
unfolding of the protein could be the cause of increased association,
consistent with the general observation that partially-unfolded protein
conformations can lead to amyloid fibril formation. (1) It should be
pointed out, however, that the Guinier region is strictly valid only for
QRg<1.3, while the above fits span Q2=0.01-0.03 Å-2
(QRg=1.7-3). Replacing [3j1(QRg)/QRg]2 with the
approximate expression exp(-Q2Rg2/3), as suggested by
Guinier, (25) results in deviations on the order of 10% over this
Q-range.

[0033]From the fits in the low-Q region of FIG. 4, the Rg-values are
approximately constant at a given pH and light condition, suggesting that
the oligomer size is primarily determined by the state of the surfactant.
I (0)-values determined from either the Guinier plots in FIG. 4 for
Q2<0.002 Å-2 or PDDFs of the overall data (not shown)
are displayed in Table 1, along with the effective oligomer size (nefo.
Together, these values suggest monomer-hexamer equilibrium for the
visible-light data and a monomer-dodecamer equilibrium under UV light,
however, unlike pure α-chymotrypsin the oligomer size is not known
a priori. Nevertheless, additional evidence will be presented below to
support this type of protein self-association. In truth, these n-mer
assignments are a result of a comprehensive iterative procedure whereby
the number of protein subunits observed from shape reconstruction of the
raw data (monomer+oligomer) were used to provide initial estimates of n.
However, since fitting the overall data gives z-averaged shape of the
protein, (42) which is heavily weighted towards the oligomer
conformation, six and 12 subunits could be consistently detected even
from the raw data (see below). Furthermore, for the UV-data, the choice
of n=12 was particularly clear given that both the SANS and SAXS data
(FIG. 8) appear to converge to an neff value of 12 with increasing
surfactant concentration.

[0034]From these resulting n-mer assignments, the monomer weight fraction
was calculated, indicating that the monomer-oligomer equilibrium shifts
towards n-mer formation with increasing surfactant concentration, a
likely result of increased partial unfolding as mentioned above. To gain
further insight in the oligomer structures, the SANS data were
deconvoluted as above to obtain the portion of the scattering due only to
the n-mers. As shown in FIG. 5, the xn-scaled oligomer scattering
data are largely consistent for a given pH and light conditions,
suggesting a sound deconvolution procedure. Some subtle changes are
observed with increasing surfactant concentration within a given data
set, particularly in the high-Q region representing fine structural
detail. Specifically, the peak observed at Q˜0.2 Å-2,
similar to the deconvoluted-hexamer of pure α-Ch at pH 7, becomes
"washed out" with increasing azoTAB concentration, suggesting that the
oligomers become more disordered with increased fluctuations in the
protein subunit positions. Using Guinier plots to calculate of the radius
of gyration from each oligomer-only scattering profile give the values of
Rgn reported in Table 1.

[0035]PDDFs calculated from the oligomer-only data display a similar
degree of homogeneity at each condition with increasing surfactant, as
shown in FIGS. 5a and 5b. Interestingly, independent of oligomer type
(hexamer or dodecamer) Rgn∝(Mw)0.42±0.03
compared to the monomer radius of gyration, where Mw is the
molecular weight of the oligomer. A similar scaling exponent (0.45) has
been reported for self-associating insulin, with values intermediate
between those expected for spheres (1/3) or Gaussian coils (1/2)
suggesting relatively open oligomer structures. (30)

[0036]Comparing the visible light (hexamer) PDDFs to FIG. 2d for the pure
hexamer reveals a shift in the PDDF peak to lower r-values. This suggests
a potential unraveling of the tightly-packed W-shaped hexamer with the
most probable dimension being reduced to distances within the protein
subunits (e.g., the protein radius) as opposed to distances between the
subunits. Although note that Dmax of the hexamer remains at
˜120 Å as in FIG. 2d, thus, only partial unraveling can be
occurring, largely retaining the twisted hexamer conformation. For
example, a linear n-mer formed from a protein with a radius of 20 Å
would give peaks at 20, 40, . . . , (n-1)40 Å. For the dodecamer
structures the most probable dimension returns to 40-50 Å, while
Dmax undergoes a modest increase to ˜160 Å, hence,
longitudinal extension of hexamers to form dodecamers does not appear to
be an appropriate mechanism. Shoulders can also be detected in the PDDF
curves at ˜80, 100, and 120 Å correspond to distances between
higher-order neighbors, suggesting regular, as opposed to random,
oligomer conformations. Guinier analyses of the oligomer-only data (not
shown) give radii of gyration of the n-mers (Rgn) consistent
with the PDDF analysis as displayed in Table 1, again largely independent
of surfactant concentration across a given data set. Taken together, this
evidence suggests that converting azoTAB to the cis form with UV light
causes hexamers to laterally (as opposed to longitudinally) associate
into dodecamers.

[0037]To obtain a better understanding of the oligomer conformations,
shape-reconstruction was applied to the deconvoluted SANS data, as shown
in FIG. 6. In all cases the shape-reconstruction algorithm returned
conformations containing either six or 12 subdomains, despite the fact
that the program begins with a random arrangement of scattering centers.
This fact further confirms the choice of hexamers and dodecamers, as well
as the overall deconvolution procedure. The shape-reconstructed hexamers
indeed support the notion above of an unraveling of the W-shaped hexamer,
as the hexamers now have extended, corkscrew-like appearances. Upon UV
illumination and conversion of the surfactant to the cis form, hexamers
are converted into the rope-like dodecamers, suggesting that dodecamer
formation result from lateral association of two hexamers. This is
illustrated by the bead-model structures accompanying each
90°-rotation view of the oligomers, used to guide the eye as to
the relative positions of each protein subunit. The observed n-mer
structures are found to be reasonably consistent across the range of pH
and surfactant concentration conditions, again pointing to the global
consistency of the deconvolution procedure.

Photo-Induced α-Chymotrypsin Oligomers are Amyloid Precursors

[0038]The lateral association of hexamers into dodecamers is consistent
with the eventual rope-like conformation commonly observed in many
amyloid fibrils, indicating that SANS may be reporting on the mechanism
of formation of key prefibrillar intermediates in the amyloid cascade. To
investigate whether the oligomer structures in FIG. 6 are true
prefibrillar intermediates, several classic amyloid tests were performed
on azoTAB/α-Ch mixtures. FT-IR spectra of pure α-Ch and
α-Ch in the presence of azoTAB under both visible and UV light are
shown in FIGS. 7a and 7b. Two aggregation processes can be triggered in
the α-Ch/azoTAB system: the first upon the addition of trans azoTAB
to pure α-Ch (dimers→hexamers at pH 3), and the second upon
exposure of the α-Ch/azoTAB system to UV light
(hexamers→dodecamers). As seen in the FT-IR spectra, both of these
association processes give rise to an increase in peaks at 1612 and 1685
cm-1, characteristic of intermolecular β-sheet formation, (5,
43, 44) at the expense of the peak at 1637 cm-1 commonly assigned to
intramolecular β-sheets. (45, 46) Zurdo et al. observe bands at 1612
and 1985 cm-1 in SH3-domain protofibril intermediates that
eventually mature into fully-developed amyloid fibrils, (44) suggesting
that the oligomers observed in FIG. 6 are indeed precursors to amyloid
structures.

[0039]The photomicrographs shown in FIGS. 7c and 7d further support this
conclusion. Congo red staining of a α-Ch/azoTAB solution aged for
five days results in characteristic Congo red fluorescence as well as
"apple green" birefringence, respectively. Congo red preferentially
stains amyloid structures due to the planar structure of the dye favoring
incorporation into the β-sheet structure of amyloids. (47-49) These
images were also accompanied by Maltese-cross patterns under cross
polarizers (not shown) indicative of spherulites formed by the aligning
of fibrils in a radial pattern. (50)

[0040]TEM images in FIG. 7 further demonstrate the formation of fibrillar
structures. FIGS. 7e and 7f were obtained two weeks after preparing a
fresh α-Ch/azoTAB solution, while FIG. 7g was obtained from an
original SANS solution (pH 3, [azoTAB]=4.2 mM) approximately one year
after collecting the SANS spectra. The fibrils shown in FIGS. 7e-g
possess clear amyloid characteristics: they are long, unbranched, and
appear to be twisted, with diameters of ca. 10 nm. Combined, these tests
confirm that the samples used in SANS are indeed pre-amyloid oligomer
intermediates.

Photo-Reversible Protein Association

[0041]To investigate photoreversible protein association, small-angle
X-ray scattering (SAXS) data were collected for mixtures of
chymotrypsinogen-A and azoTAB at pH 3, as shown in FIG. 8.
Chymotrypsinogen is the zymogen of α-chymotrypsin, activated by the
removal of two dipeptides at positions Ser14-ArglS and Thr147-Asn148
leading to the formation of the active site. (51) Despite this structural
similarity, however, chymotrypsinogen does not generally associate in
solution unlike in the case of α-chymotrypsin. (52, 53)

[0042]This phenomena is supported by the visible-light SAXS data in FIG.
8a, where a clear intermolecular interaction peak is observed in contrast
to FIG. 1, consistent with increasing electrostatic repulsion between
chymotrypsinogen monomers as the cationic surfactant binds to the
positively-charged protein. A Guinier plot of the pure-protein SAXS data
(FIG. 8 inset) gives Rg=17.1 Å, similar to the SAXS-derived
Rg-value from the literature of 17.6 Å. (39) With increasing
surfactant concentration, Rg increases modestly up to 10 mM azoTAB
under visible light, eventually increasing to 19.7 Å at 24 mM azoTAB
(Table 2). The enhanced negative deviations from the Guinier behavior at
low Q with increasing surfactant concentration are a result of increasing
intermolecular interactions.

[0043]Under UV light illumination, however, the situation is markedly
different, with large increases in the SAXS data observed at low Q (note
that the y-axes of FIGS. 8a and 8b differ by an order of magnitude),
particularly at 10 mM azoTAB and beyond, coincidentally the surfactant
concentration where the onset of chymotrypsinogen unfolding was observed
under visible light. The Guinier plots under UV light also reveal the
development of an additional larger species with increasing surfactant
concentration, detected by the appearance of a steep slope at low Q. I(0)
values for samples under visible light (˜0.2-0.25 cm-1, see
Table 2, where the SAXS data have been put on absolute scale by comparing
to a calibration standard of 10 mg/mL BSA (10)) are consistent with the
value expected for the monomer (I(0)=0.24 cm-1), again indicating
that chymotrypsinogen association does not occur under visible light.
Under UV light a 12-fold increase in I(0) is observed at 19 mM and 24 mM
azoTAB relative to the monomer data, suggesting that the association
equilibrium is pushed entirely towards dodecamers, providing independent
confirmation of the α-chymotrypsin data.

[0044]Reversibility of protein self-association is shown in FIG. 8b, where
SAXS spectra were collected for UV-equilibrated samples following
re-exposure to 434-nm visible light. The low-Q scattering decreases as a
function of visible-light exposure time, with apparently several hours
required for complete visible-light induced dissociation (beyond the
limit of the allocated SAXS beam time). However, it should be pointed out
that this dissociation process is not limited by the cis→trans
isomerization kinetics, which occurs within minutes (20). Protein
association and dissociation can generally occur on time scales ranging
from seconds up to hours or even several days. (54, 55) Thus, the SAXS
data demonstrate photoreversible control of protein oligomerization.

[0045]Obviously, many modifications and variation of the invention as
hereinbefore set forth can be made without departing from the spirit and
scope thereof and therefore only such limitations should be imposed as
are indicated by the appended claims.

[0046]All patent and literature references cited in the present
specification are hereby incorporated by reference in their entirety.